The proper targeting of proteins to either the apical or basolateral surface of polarized airway epithelial cells is critical for normal lung function. An important example is cystic fibrosis, a disease caused in large part by a single amino acid mutation in the CFTR Cl- channel that prevents its transport to the apical surface of airway cells. Other important proteins, such as G protein-coupled P2Y receptors for extracellular nucleotides, are expressed in airway and other polarized epithelial cells and are targeted to different membrane domains of these cells. Thus, the P2Y1 receptor is expressed exclusively at the basolateral surface of polarized epithelial cells, whereas the P2Y2 receptor is expressed at the apical surface. Very little is known about the sorting signals and mechanisms by which G protein-coupled receptors in general, and P2Y receptors in particular, are targeted to their final destination in polarized cells. In this application, we propose to identify the mechanisms by which P2Y1 and P2Y2 receptors are targeted to opposite surfaces of airway and other polarized epithelial cells. We will first establish the routes by which P2Y1 and P2Y2 receptors are delivered to the basolateral and apical domains of polarized cells. In the second aim, we will identify the targeting domains that direct polarized expression of the two receptors by utilizing both confocal microscopy and quantification of cell-surface expression in MDCK cells. We will delimit the regions that are responsible for polarized expression of the receptors, define the amino acids that are most critical, and determine whether the sorting signals direct targeting of non-sorted receptors. Finally, we will identify proteins that interact with the targeting domains of these receptors by yeast 2-hybrid and proteomics approaches. Once identified, we will characterize these proteins to determine if they direct polarized targeting. These experiments will provide important information into the mechanism by which P2Y receptors achieve their steady-state localization in polarized epithelial cells and generate insights into how G protein-coupled receptors in general are targeted to different membrane domains.